This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The centrosome plays a fundamental role in organizing the microtubule cytoskeleton. Defects in centrosome function lead to errors in chromosome segregation, a major factor in both the initiation and progression of cancer. We aim to understand the mechanism and regulation of microtubule nucleation at the centrosome by focusing on the structure of a key 300 kDa heterotetrameric complex that underlies nucleation in all eukaryotes: the [unreadable]-tubulin small complex ([unreadable]-TuSC). We have determined a negative stain EM structure by the random conical tilt method, and have preliminary images under cryo conditions. From a higher resolution cryo-EM structure we hope to determine the overall organization of the complex and particularly which surfaces of [unreadable]-tubulin are exposed for interaction with [unreadable][unreadable]-tubulin at the microtubule minus ends, and to map the binding sites of proteins which link [unreadable]-TuSC to microtubule organizing centers.